Immunoglobulin G (IgG) Receptors (Fcxcex3R)
An IgG antibody molecule has two binding determinants (known as Fab) for its specific cognate antigen, and one binding determinant (Fc) for its cellular receptor (Fcxcex3R; reviewed in Ravetch, J. V. and J-P. Kinet, 1991, Ann. Rev. Imm. 9:457). There are three classes of Fcxcex3R, designated Fcxcex3RI, Fcxcex3RII and Fcxcex3RIII. Fcxcex3RI has the highest affinity for IgG Fc, and is present on monocytes and macrophages, and on human neutrophils when induced by gamma-interferon (IFN-xcex3), which also enhances Fcxcex3RI expression on monocytes and macrophages (Guyre, P. M. et al., 1983, J.Clin.Investig. 72:393). Interleukin-10 (IL-10) is also a potent up-regulator of Fcxcex3RI expression on monocytes (Capsoni, F. et al., 1995, J. Leukoc. Biol. 58:351). Neutrophil expression of Fcxcex3RI is increased almost four-fold by granulocyte-colony stimulating factor (G-CSF; Repp, R. et al., 1991, Blood 78:885), and about three-fold during streptococcal pharyngitis (Guyre, P. M. et al., 1990, J. Clin. Investig. 86:1892). G-CSF stimulation is associated with enhanced neutrophil-mediated cytotoxicity of tumor cells (Gosselin, E. et al., 1992, J. Immunol. 149:3477).
Cellular distribution of the low affinity receptor Fcxcex3RII is broader, extending to neutrophils, platelets, B cells, T cell, and possibly mast cells and basophils, in addition to monocytes and macrophages. Human Fcxcex3RIII receptors are restricted to macrophages and macrophage cell lines, natural killer (NK) cells, myeloid precursor cells, and a neutrophil cell line (Ravetch and Kinet, supra).
The interaction of antibody-antigen complexes with cells via Fcxcex3R triggers a range of responses including antibody-dependent cytotoxicity, degranulation, phagocytosis, and immunomodulatory signals that regulate lymphocyte proliferation and antibody secretion. Fcxcex3RI, for example, is active in enhancing antigen presentation (Valerius, T. et al., 1993, Blood 82:931) and the Fcxcex3RI complementarity designation CD64 has been used to distinguish granulomonocytic progenitor cell populations within a human progenitor cell mixture (CD34+/CD38+; European Patent Application EP O 708 336 A2).
Further, a monoclonal antibody (mAb) that binds Fcxcex3RI has been shown to cause down-regulation of expression of this receptor, and administration of this mAb to a chronic immune thrombocytopenia purpura patient resulted in clinical improvement (Ericson, S. G., 1996, Brit. J. Haematol. 92:718). The analysis of in vivo function of Fcxcex3RI receptors is complicated by the finding that four healthy members of one family lack Fcxcex3RI (Ceuppens, J. L., 1988, J. Clin. Invest. 82:571).
Cell Staining with PE-Cy5
The tandem dye PE-Cy5 comprises the combination of two fluorescent dyes, R-phycoerythrin (PE) and Cy5.18.OSu (Cy5; Mujumdar, R. B. et al., 1993, Bioconj. Chem. 4:105). PE-Cy5 is commonly used conjugated to a monoclonal antibody (mAb), as described in for example, the Jan. 1, 1996, issue of Blood that reviews the structure and biology of CD34, accompanied by a cover photograph of a flow cytometric experiment with PE-Cy5 conjugated to an anti-CD34 mAb (Krause D S, et al., 1996, Blood 87:1). PE-Cy5 tandem dye is known as xe2x80x9ctricolorxe2x80x9d since it is frequently used with other dye-mAb conjugates for three-color flow cytometric analyses (Waggoner A S, et al., In: Clinical Flow Cytometry, p.185 (Eds) A. Landay et al. The New York Academy of Sciences, New York, N.Y., 1993).
A non-toxic molecule that binds specifically to Fcxcex3RI in vivo would be useful in a variety of diagnostic and therapeutic applications.
In general the invention relates to agents that specifically bind IgG receptors of the class Fcxcex3RI on white blood cells (leukocytes). Because of the functional binding of these compounds, the class of compositions is referred to herein as xe2x80x9cGRILxe2x80x9d to indicate an IgG receptor I ligand. The agents are members of the cyanidin family of naturally-occurring plant pigments, exemplified by cyanin, idaein, keracyanin and asterin.
In one aspect, the invention features a class of cyanin compositions of which the fluorescent dye Cy5.18.OSu (referred to as Cy5), and conjugates and derivatives, are examples. The general features of these compositions are firstly functional, which is that they bind with high affinity and specificity to Fcxcex3RI IgG receptors found on monocytes and macrophages, and which can also be induced to appear on neutrophils. The second feature is the chemical structure, which may be comprised of at least two moieties, one of which being the cyanin succinimidyl ester composition class. In one embodiment, the second moiety is derived from any of the phycobilisome proteins of which PE is an exemplary but not limiting member.
In another aspect, the present invention features therapeutic compositions comprising a GRIL, for example, a cyanin compound exemplified by a Cy5 reagent, and a therapeutic agent, which therapeutic agent in a preferred embodiment comprises at least one epitope. In one embodiment, the GRIL directs delivery of the useful epitope to Fcxcex3RI, and thus comprises a composition which is both vaccine and delivery system. The epitope can be selected from the broad group consisting of a pathogenic organism, a cancer marker, and an allergenic substance. The compounds can thus be used to vaccinate a subject against a pathogenic organism, such as a bacterium, a protozoan, a virus or a fungus. In a preferred embodiment, the epitope for a vaccine is from a Gram positive pathogen, for example, from a cellular or secreted epitope of Staphyloccoccus aureus or Clostridium tetani. The invention also features a vaccine against one or more types of cancer, comprising a GRIL and an epitope from a cancer marker which, in a preferred embodiment, is selected from the group consisting of the human EGF receptor family, the mucine TAG72 family, and the carcinoembryonic family.
In another aspect, the present invention features therapeutic compositions comprising a GRIL and a chemotherapeutic cytotoxic anticancer agent, for example, actinomycin D, mitomycin C, adriamycin, taxol, notirimine, or a therapeutic composition comprising a Cy5 reagent and a radionuclide. The therapeutic agent is thus targetted to Fcxcex3RI by virtue of the presence of the cyanin, for example a Cy5 or similar reagent, and is useful for treatment of, for example, a myelocytic leukemia, a promyelocytic leukemia or a leukocyte adhesion deficiency.
Methods for targetting Fcxcex3RI receptors with a chemotherapeutic or radiotherapeutic GRIL is another aspect of this invention, for the purpose of treating for example, a patient with an autoimmune disease, such as idiopathic thrombocytopenic purpura. Since the agents of the instant invention target IgG receptors, which are located on white blood cells, the methods of the invention include delivery of these compositions ex vivo to cells obtained from a blood sample or a bone marrow sample removed from the patient for such treatment, which blood or bone marrow cell sample or its cellular progeny is subsequently returned to that patient. Further, an interferon, an interleukin or a colony stimulating factor may also be administered to the patient or to the ex vivo blood or bone marrow sample or to cells derived from such samples. In preferred embodiments, the patient is administered IFN-xcex3, IL-10, or G-CSF.
Another aspect of the invention features compositions and diagnostic methods for detecting and quantifying Fcxcex3RI (CD64) in a blood sample or a bone marrow sample from a subject, comprising the steps of contacting the sample with a GRIL stain, removing nonspecifically-bound reagent, illuminating the sample with light of an appropriate excitation wavelength, and detecting the fluorescent cells as an indication of Fcxcex3RI-bearing cells in the blood sample. Preferably, at least one additional dye reagent, for example, a fluorescein reagent or a phycoerythrin reagent is used in conjunction with the GRIL. In a preferred embodiment, the GRIL-stained cells are analyzed and isolated by flow cytometry. In another embodiment, the GRIL-stained cells are analyzed by fluorescent microscopy. In these embodiments, the excitation wavelength for illumination is preferably in the range of approximately 475 to 505 nm. Most preferably, the number of Fcxcex3RI in blood is an index of the diagnosis, which is measured under appropriate conditions when the detection method is used diagnostically. In a preferred embodiment, the method is used as a diagnostic for leukocyte adhesion deficiency or for a myeloid or promyeloid leukemia; for the presence of infection and/or inflammation; or to monitor therapy during and following administration of a therapeutic agent. The therapeutic agent is preferably an interferon, an interleukin, or a colony stimulating factor, particularly IFN-xcex3, IL-10, or G-CSF.
In yet another aspect, the invention features a method for screening compounds and identifying among them candidate therapeutic agents which bind to Fcxcex3RI. This method comprises contacting a first sample of cells with a candidate agent and contacting a second sample of cells with control buffer, then staining both the first and second samples of cells with a GRIL stain. The samples are illuminated with a wavelength appropriate to cause fluorescence of cells with specifically bound dye, and the fluorescence is used as an index of stained cells, such that an agent which reduces staining of the first sample in comparison to the second sample to a statistically significant extent is a potential therapeutic candidate. The method preferably also comprises a further step of formulating a pharmaceutical preparation of each of the compounds identified by this method as having potential therapeutic activity. This method enables the user to identify low molecular weight drug candidates that interact with IgG receptors. Such drug candidates might be useful in modulation of Fcxcex3RI expression, or in preventing Fc binding to Fcxcex3RI.
The invention features also a kit for assaying Fcxcex3RI on cells, comprising a GRIL stain, a buffer, a container, and instructions for use. This kit is useful for diagnostic purposes. Another kit embodied by the invention is useful for blocking adsorption of cyanidin and Cy5 reagents to Fcxcex3RI, comprising an anti-Fcxcex3RI antibody, a GRIL stain reagent, a buffer, a container, and instructions for use. This kit would block binding of GRIL reagents to Fcxcex3RI, so that such reagents could be used to stain other cell surface molecules.
Other features and advantages of the instant invention will become more apparent form the following detailed description and claims.